Phospholipid-based powders for drug delivery

ABSTRACT

Phospholipid based powders for drug delivery applications are disclosed. The powders may include a polyvalent cation in an amount effective to increase the gel-to-liquid crystal transition temperature of the particle compared to particles without the polyvalent cation. The powders are hollow and porous and are preferably administered via inhalation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/851,226, filed on May 8, 2001, now U.S. Pat. No. 7,442,388,which is a continuation-in-part of U.S. patent application Ser. No.09/568,818, filed May 10, 2000, and is also a continuation applicationof U.S. patent application Ser. No. 10/141,219, filed on May 7, 2002,both of which claim benefit of the priority of U.S. ProvisionalApplication Ser. No. 60/208,896 filed Jun. 2, 2000 and U.S. ProvisionalApplication Ser. No. 60/216,621 filed Jul. 7, 2000.

FIELD OF THE INVENTION

The present invention relates to particulate compositions suitable fordrug delivery, preferably via inhalation. In particular, the presentinvention provides phospholipid-containing particulate compositionscomprising a polyvalent cation. The particulate compositions of thepresent invention exhibit an increased gel-to-liquid crystal transitiontemperatures resulting in improved dispersibility and storage stability.

BACKGROUND OF THE INVENTION

Phospholipids are major components of cell and organelle membranes,blood lipoproteins, and lung surfactant. In terms of pulmonary drugdelivery, phospholipids have been investigated as therapeutic agents forthe treatment of respiratory distress syndrome (i.e. exogenous lungsurfactants), and as suitable excipients for the delivery of actives.The interaction of phospholipids with water is critical to theformation, maintenance, and function of each of these importantbiological complexes (McIntosh and Magid). At low temperatures in thegel phase, the acyl chains are in a conformationally well-ordered state,essentially in the all-trans configuration. At higher temperatures,above the chain melting temperature, this chain order is lost, owing toan increase in gauche conformer content (Seddon and Cevc).

Several exogenous lung surfactants have been marketed and includeproducts derived from bovine lungs (Survanta®, Abbott Laboratories),porcine lungs (CuroSurf®, Dey Laboratories), or completely syntheticsurfactants with no apoproteins (e.g. ALEC®, ExoSurf® Glaxo Wellcome).To date, these products have been utilized for the treatment of infantrespiratory distress syndrome (IRDS). None have been successful inreceiving FDA approval for the treatment of adult respiratory distresssyndrome (ARDS). The current infant dose is 100 mg/kg. For a 50 kgadult, this would translate into a dose of 5 g. A dose of this amountcan only be administered to ARDS patients by direct instillation intothe patient's endotracheal tube, or possibly via nebulization of aqueousdispersions of the surfactant material.

Instillation of surfactants leads to deposition primarily in the centralairways, and little of the drug makes it to the alveoli, where it isneeded to improve gas exchange in these critically ill patients.Nebulization of surfactant may allow for greater peripheral delivery,but is plagued by the fact that (a) current nebulizers are inefficientdevices and only ca. 10% of the drug actually reaches the patientslungs; (b) the surfactant solutions foam during the nebulizationprocess, leading to complications and further loss of drug. It isbelieved that as much as 99% of the administered surfactant may bewasted due to poor delivery to the patient. If more effective deliveryof surfactant could be achieved, it is likely that the administered doseand cost for treatment of ARDS could be dramatically decreased.

Further, lung surfactant has been shown to modulate mucous transport inairways. In this regard, the chronic administration of surfactant forthe treatment of patients with chronic obstructive pulmonary disease(COPD) has been suggested. Still other indications with significantlylower doses may be open to treatment if a dry powder form of a lungsurfactant were available. The powdered surfactant formulation may bepurely synthetic (i.e. with no added apoproteins). Alternatively, thepowder formulation could contain the hydrophobic apoproteins SP-B orSP-C or alternative recombinant or synthetic peptide mimetics (e.g.KL₄).

Due to its spreading characteristics on lung epithelia, surfactant hasbeen proposed as the ideal carrier for delivery of drugs to the lung,and via the lung to the systemic circulation. Once again, achievingefficient delivery to the lung is important, especially in light of thepotential high cost of many of the current products. One potential wayto deliver drugs in phospholipids is as a dry powder aerosolized to thelung. Most fine powders (<5 μm) exhibit poor dispersibility. This can beproblematic when attempting to deliver, aerosolize, and/or package thepowders.

The major forces that control particle-particle interactions can bedivided into short and long range forces. Long-range forces includegravitational attractive forces and electrostatics, where theinteraction varies as the square of the separation distance. Short-rangeattractive forces dominate for dry powders and include van der Waalsinteractions, hydrogen bonding, and liquid bridging. Liquid bridgingoccurs when water molecules are able to irreversibly bind particlestogether.

Phospholipids are especially difficult to formulate as dry powders astheir low gel to liquid crystal transition temperature (Tm) values andamorphous nature lead to powders which are very sticky and difficult todeaggregate and aerosolize. Phospholipids with Tm values less than 10°C. (e.g. egg PC or any unsaturated lipids) form highly cohesive powdersfollowing spray-drying. Inspection of the powders via scanning electronmicroscopy reveals highly agglomerated particles with surfaces thatappear to have been melted/annealed. Formulating phospholipid powderswhich have low Tm are problematic, especially if one hopes to achieve acertain particle morphology, as in the case of aerosol delivery. Thus,it would be advantageous to find ways to elevate the Tm of these lipids.Examples of particulate compositions incorporating a surfactant aredisclosed in PCT publications WO 99/16419, WO 99/38493, WO 99/66903, WO00/10541, and U.S. Pat. No. 5,855,913, which are hereby incorporated intheir entirety by reference.

Currently, lung surfactant is given to patients by intubating them andinstilling a suspension of lung surfactant directly into the lungs. Thisis a highly invasive procedure which generally is not performed onconscious patients, and as do most procedures, carries its own risks.Potential applications for lung surfactant beyond the current indicationof respiratory distress syndrome in neonates are greatly limited by thismethod of administration. For example, lung surfactant may be useful ina variety of disease states that are, in part, due to decreased lungsurfactant being present in the lungs. U.S. Pat. Nos. 5,451,569,5,698,537, and 5,925,337, and PCT publications WO 97/26863 and WO00/27360, for example, disclose the pulmonary administration of lungsurfactant to treat various conditions, the disclosures of which arehereby incorporated in their entirety by reference. Diseases that arethought to be possibly aggravated by lung surfactant deficiency includecystic fibrosis, chronic obstructive pulmonary disease, and asthma, justto name a few. The delivery of exogenous lung surfactant, in a topicalfashion, to patients suffering from these diseases may amelioratecertain signs and symptoms of the diseases. For chronic conditions, theregular (once or more times per day on a prolonged basis) delivery oflung surfactant via intubation and instillation to ambulatory patientsis impractical. Further, because of their high surface activity, lungsurfactant suspensions are not amenable to nebulization due to foaming.The current delivery of phospholipid-based preparations by instillationor nebulization are highly inefficient in delivering material to theperipheral lung. Therefore, the ability to deliver lung surfactant topatients via dry powder inhalation would be a tremendous advantage overthe current method, since it would avoid the need for intubation,thereby expanding the potential uses of lung surfactant in the clinicalsetting.

SUMMARY OF THE INVENTION

The present invention provides for dry powder compositions ofphospholipid suitable for drug delivery. According to a preferredembodiment, the phospholipid compositions are efficiently delivered tothe deep lung. The phospholipid may be delivered alone, as in the caseof lung surfactant or in combination with another active agent and/orexcipient. The use of dry powder compositions may also open newindications for use since the patient need not be intubated. Accordingto one embodiment, the compositions of the present invention may bedelivered from a simple passive DPI device. The present compositionsallow for greater stability on storage, and for more efficient deliveryto the lung.

It has been found in the present work that the gel to liquid crystalphase transition of the phospholipid, Tm, is critical in obtainingphospholipid-based dry powders that both flow well, and are readilydispersible from a dry powder inhaler device. The present invention isrelated to the use of polyvalent cations, preferably divalent cations todramatically increase the Tm of phospholipids. As used herein,“polyvalent cations” refers to polyvalent salts or their ioniccomponents. Increasing the Tm of the phospholipid leads to the followingformulation improvements: (a) Increases in Tm allows the formulator toincrease the inlet and outlet temperatures on the spray-drier, or on avacuum oven during a secondary drying step. Higher temperatures allowthe drying phase of the spray-drying to be controllable over a widertemperature range, thereby facilitating removal of trapped blowing agentused in the manufacture of powders according to one aspect of thepresent invention; (b) Increases in Tm allow for a large differencebetween Tm and the storage temperature, thereby improving powderstability; (c) Increases in Tm yield phospholipids in the gel state,where they are less prone to taking up water and water bridgingphenomena (d) Increases in Tm yield phospholipids which are able tospread more effectively upon contact with lung epithelia than hydratedphospholipids, thereby allowing drugs to be more effectively distributedto the lung periphery; (e) Increases in Tm dramatically improves thedispersibility of the resulting powders, thereby improving the emitteddose and fine particle fraction following pulmonary delivery.

According to a preferred embodiment, the present invention relates tohighly dispersible dry powder compositions of phospholipids suitable forpulmonary delivery. The compositions according to the present inventionare useful as synthetic lung surfactants for the treatment of local lungconditions (e.g. asthma, COPD), or as carriers for the pulmonarydelivery of active agents, including small molecules, peptides,proteins, DNA, and immunologic agents.

One aspect of the present invention is to provide powdered, dispersiblecompositions having stable dispersibility over time. The compositionsexhibit a characteristic gel to liquid crystal phase transitiontemperature, Tm, which is greater than a recommended storagetemperature, Ts, typically room temperature, by at least 20° C.Preferably Tm is at least 40° C. greater than Ts.

It is a further aspect of the present invention that the increases in Tmafforded by addition of divalent cations leads to the ability to dry thepowders in a secondary drying step at temperatures (Td) up to the Tm ofthe lipid. As well, it is possible to increase the inlet and outlettemperatures on a spray-drier should a spray-dry process be employed(Td≈Tm).

It is a further aspect of the present invention to provide a powdered,dispersible form of a lung surfactant having stable dispersibility overtime and excellent spreading characteristics on an aqueous subphase.

It is a further aspect of the present invention that the improvements indispersibility obtained by the present compositions allow for a simple,passive inhaler device to be utilized, in spite of the fact thatparticles less than 5 μm are contemplated and generally preferred.Present state-of-the-art formulations for fine particles utilize blendswith large lactose particles to improve dispersibility. When placed in apassive DPI device such formulations exhibit a strong dependence ofemitted dose and lung deposition on the patient's inspiratory flowrate.The present compositions exhibit little flowrate dependence on theemitted dose and lung deposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the physical stability of budesonide inpMDI.

FIG. 2 are SEM photographs the effect of calcium ion concentration onthe morphology of spray-dried particles according to the invention.

FIG. 3 is a graph depicting the spreading characteristics of powders ofthe instant invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Active agent” as described herein includes an agent, drug, compound,composition of matter or mixture thereof which provides some diagnostic,prophylactic, or pharmacologic, often beneficial, effect. This includesfoods, food supplements, nutrients, drugs, vaccines, vitamins, and otherbeneficial agents. As used herein, the terms further include anyphysiologically or pharmacologically active substance that produces alocalized or systemic effect in a patient. The active agent that can bedelivered includes antibiotics, antibodies, antiviral agents,anepileptics, analgesics, anti-inflammatory agents and bronchodilators,and viruses and may be inorganic and organic compounds, including,without limitation, drugs which act on the peripheral nerves, adrenergicreceptors, cholinergic receptors, the skeletal muscles, thecardiovascular system, smooth muscles, the blood circulatory system,synaptic sites, neuroeffector junctional sites, endocrine and hormonesystems, the immunological system, the reproductive system, the skeletalsystem, autacoid systems, the alimentary and excretory systems, thehistamine system and the central nervous system. Suitable agents may beselected from, for example, polysaccharides, steroids, hypnotics andsedatives, psychic energizers, tranquilizers, anticonvulsants, musclerelaxants, antiparkinson agents, analgesics, anti-inflammatories, musclecontractants, antimicrobials, antimalarials, hormonal agents includingcontraceptives, sympathomimetics, polypeptides, and proteins capable ofeliciting physiological effects, diuretics, lipid regulating agents,antiandrogenic agents, antiparasitics, neoplastics, antineoplastics,hypoglycemics, nutritional agents and supplements, growth supplements,fats, antienteritis agents, electrolytes, vaccines and diagnosticagents.

Examples of active agents useful in this invention include but are notlimited to insulin, calcitonin, erythropoietin (EPO), Factor VIII,Factor IX, ceredase, cerezyme, cyclosporine, granulocyte colonystimulating factor (GCSF), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), growthhormone, human growth hormone (hGH), growth hormone releasing hormone(GHRH), heparin, low molecular weight heparin (LMWH), interferon alpha,interferon beta, interferon gamma, interleukin-2, luteinizing hormonereleasing hormone (LHRH), leuprolide, somatostatin, somatostatin analogsincluding octreotide, vasopressin analog, follicle stimulating hormone(FSH), immunoglobulins, insulin-like growth factor, insulintropin,interleukin-1 receptor antagonist, interleukin-3, interleukin-4,interleukin-6, macrophage colony stimulating factor (M-CSF), nervegrowth factor, parathyroid hormone (PTH), thymosin alpha 1, IIb/IIIainhibitor, alpha-1 antitrypsin, respiratory syncytial virus antibody,cystic fibrosis transmembrane regulator (CFTR) gene, deoxyribonuclease(Dnase), bactericidal/permeability increasing protein (BPI), anti-CMVantibody, interleukin-1 receptor, 13-cis retinoic acid, nicotine,nicotine bitartrate, gentamicin, ciprofloxacin, amphotericin, amikacin,tobramycin, pentamidine isethionate, albuterol sulfate, metaproterenolsulfate, beclomethasone dipropionate, triamcinolone acetamide,budesonide acetonide, ipratropium bromide, flunisolide, fluticasone,fluticasone propionate, salmeterol xinofoate, formeterol fumarate,cromolyn sodium, ergotamine tartrate and the analogues, agonists andantagonists of the above. Active agents may further comprise nucleicacids, present as bare nucleic acid molecules, viral vectors, associatedviral particles, nucleic acids associated or incorporated within lipidsor a lipid-containing material, plasmid DNA or RNA or other nucleic acidconstruction of a type suitable for transfection or transformation ofcells, particularly cells of the alveolar regions of the lungs. Theactive agents may be in various forms, such as soluble and insolublecharged or uncharged molecules, components of molecular complexes orpharmacologically acceptable salts. The active agents may be naturallyoccurring molecules or they may be recombinantly produced, or they maybe analogs of the naturally occurring or recombinantly produced activeagents with one or more amino acids added or deleted. Further, theactive agent may comprise live attenuated or killed viruses suitable foruse as vaccines.

As used herein, the term “emitted dose” or “ED” refers to an indicationof the delivery of dry powder from a suitable inhaler device after afiring or dispersion event from a powder unit or reservoir. ED isdefined as the ratio of the dose delivered by an inhaler device(described in detail below) to the nominal dose (i.e., the mass ofpowder per unit dose placed into a suitable inhaler device prior tofiring). The ED is an experimentally-determined amount, and is typicallydetermined using an in-vitro device set up which mimics patient dosing.To determine an ED value, a nominal dose of dry powder (as definedabove) is placed into a suitable dry powder inhaler, which is thenactuated, dispersing the powder. The resulting aerosol cloud is thendrawn by vacuum from the device, where it is captured on a tared filterattached to the device mouthpiece. The amount of powder that reaches thefilter constitutes the delivered dose. For example, for a 5 mg, drypowder-containing blister pack placed into an inhalation device, ifdispersion of the powder results in the recovery of 4 mg of powder on atared filter as described above, then the ED for the dry powdercomposition is: 4 mg (delivered dose)/5 mg (nominal dose)×100=80%.

“Mass median diameter” or “MMD” is a measure of mean particle size,since the powders of the invention are generally polydisperse (i.e.,consist of a range of particle sizes). MMD values as reported herein aredetermined by laser diffraction, although any number of commonlyemployed techniques can be used for measuring mean particle size.

“Mass median aerodynamic diameter” or “MMAD” is a measure of theaerodynamic size of a dispersed particle. The aerodynamic diameter isused to describe an aerosolized powder in terms of its settlingbehavior, and is the diameter of a unit density sphere having the samesettling velocity, generally in air, as the particle. The aerodynamicdiameter encompasses particle shape, density and physical size of aparticle. As used herein, MMAD refers to the midpoint or median of theaerodynamic particle size distribution of an aerosolized powderdetermined by cascade impaction.

The present invention is directed to the formulation of dryphospholipid-polyvalent cation based particulate composition. Inparticular, the present invention is directed to the use of polyvalentcations in the manufacture of phospholipid-containing, dispersibleparticulate compositions for pulmonary administration to the respiratorytract for local or systemic therapy via aerosolization, and to theparticulate compositions made thereby. The invention is based, at leastin part, on the surprising discovery of the beneficial aerosolizationand stabilization properties of phospholipid-containing particulatecompositions comprising a polyvalent cation. These unexpected benefitsinclude a dramatic increase in the gel-to-liquid crystal phasetransition temperature (Tm) of the particulate composition, improveddispersibility of such particulate compositions, improved spreadabilityof the particulate compositions upon contact with lung epithelia therebyallowing drugs to be more effectively distributed to the lung periphery,and improved storage stability of the particulate compositions.

It is surprisingly unexpected that the addition of a very hygroscopicsalt such as calcium chloride would stabilize a dry powder prone tomoisture induced destabilization, as one would expect that the calciumchloride would readily pick up water leading to particle aggregation.However, this is not what is observed. In contrast, addition of calciumions leads to a dramatic improvement in the stability of the dryphospholipid-based powder to humidity. While not being bound to anytheory, it is believed that calcium ions are believed to intercalate thephospholipid membrane, thereby interacting directly with the negativelycharged portion of the zwitterionic headgroup. The result of thisinteraction is increased dehydration of the headgroup area andcondensation of the acyl-chain packing, all of which leads to increasedthermodynamic stability of the phospholipids.

The polyvalent cation for use in the present invention is preferably adivalent cation including calcium, magnesium, zinc, iron, and the like.According to the invention, the polyvalent cation is present in anamount effective to increase the Tm of the phospholipid such that theparticulate composition exhibits a Tm which is greater than its storagetemperature Ts by at least 20° C., preferably at least 40° C. The molarratio of polyvalent cation to phospholipid should be at least 0.05,preferably 0.05-2.0, and most preferably 0.25-1.0. A molar ratio ofpolyvalent cation:phospholipid of about 0.50 is particularly preferredaccording to the present invention. Calcium is the particularlypreferred polyvalent cation of the present invention and is provided ascalcium chloride.

In a broad sense, phospholipid suitable for use in the present inventioninclude any of those known in the art.

According to a preferred embodiment, the phospholipid is most preferablya saturated phospholipid. According to a particularly preferredembodiment, saturated phosphatidylcholines are used as the phospholipidof the present invention. Preferred acyl chain lengths are 16:0 and 18:0(i.e. palmitoyl and stearoyl). According to one embodiment directed tolung surfactant compositions, the phospholipid can make up to 90 to99.9% w/w of the composition. Suitable phospholipids according to thisaspect of the invention include natural or synthetic lung surfactantssuch as those commercially available under the trademarks ExoSurf,InfaSurf® (Ony, Inc.), Survanta, CuroSurf, and ALEC. For drug deliverypurposes wherein an active agent is included with the particulatecomposition, the phospholipid content will be determined by the drugactivity, the mode of delivery, and other factors and will likely be inthe range from about 20% to up to 99.9% w/w. Thus, drug loading can varybetween about 0.1% and 80% w/w, preferably 5-70% w/w.

According to a preferred embodiment, it has been found in the presentwork that the Tm of the phospholipid is critical in obtainingphospholipid-based dry powders that both flow well and are readilydispersible from a dry powder inhaler (DPI). The Tm of the modifiedlipid microparticles can be manipulated by varying the amount ofpolyvalent cations in the formulation.

Phospholipids from both natural and synthetic sources are compatiblewith the present invention and may be used in varying concentrations toform the structural matrix. Generally compatible phospholipids comprisethose that have a gel to liquid crystal phase transition greater thanabout 40° C. Preferably the incorporated phospholipids are relativelylong chain (i.e. C₁₆-C₂₂) saturated lipids and more preferably comprisesaturated phospholipids, most preferably saturated phosphatidylcholineshaving acyl chain lengths of 16:0 or 18:0 (palmitoyl and stearoyl).Exemplary phospholipids useful in the disclosed stabilized preparationscomprise, phosphoglycerides such as dipalmitoylphosphatidylcholine,disteroylphosphatidylcholine, diarachidoylphosphatidylcholinedibehenoylphosphatidylcholine, diphosphatidyl glycerol, short-chainphosphatidylcholines, long-chain saturated phosphatidylethanolamines,long-chain saturated phosphatidylserines, long-chain saturatedphosphatidylglycerols, long-chain saturated phosphatidylinositols.

In addition to the phospholipid, a co-surfactant or combinations ofsurfactants, including the use of one or more in the liquid phase andone or more associated with the particulate compositions arecontemplated as being within the scope of the invention. By “associatedwith or comprise” it is meant that the particulate compositions mayincorporate, adsorb, absorb, be coated with or be formed by thesurfactant. Surfactants include fluorinated and nonfluorinated compoundsand are selected from the group consisting of saturated and unsaturatedlipids, nonionic detergents, nonionic block copolymers, ionicsurfactants and combinations thereof. In those embodiments comprisingstabilized dispersions, such nonfluorinated surfactants will preferablybe relatively insoluble in the suspension medium. It should beemphasized that, in addition to the aforementioned surfactants, suitablefluorinated surfactants are compatible with the teachings herein and maybe used to provide the desired preparations.

Compatible nonionic detergents suitable as co-surfactants comprise:sorbitan esters including sorbitan trioleate (Span™ 85), sorbitansesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene(20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate,oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether,lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters.Other suitable nonionic detergents can be easily identified usingMcCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock,N.J.) which is incorporated herein in its entirety. Preferred blockcopolymers include diblock and triblock copolymers of polyoxyethyleneand polyoxypropylene, including poloxamer 188 (Pluronic™ F-68),poloxamer 407 (Pluronic™ F-127), and poloxamer 338. Ionic surfactantssuch as sodium sulfosuccinate, and fatty acid soaps may also beutilized.

Other lipids including glycolipids, ganglioside GM1, sphingomyelin,phosphatidic acid, cardiolipin; lipids bearing polymer chains such aspolyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone;lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acidssuch as palmitic acid, stearic acid, and oleic acid; cholesterol,cholesterol esters, and cholesterol hemisuccinate may also be used inaccordance with the teachings of this invention.

It will further be appreciated that the particulate compositionsaccording to the invention may, if desired, contain a combination of twoor more active ingredients. The agents may be provided in combination ina single species of particulate composition or individually in separatespecies of particulate compositions. For example, two or more activeagents may be incorporated in a single feed stock preparation and spraydried to provide a single particulate composition species comprising aplurality of active agents. Conversely, the individual actives could beadded to separate stocks and spray dried separately to provide aplurality of particulate composition species with differentcompositions. These individual species could be added to the suspensionmedium or dry powder dispensing compartment in any desired proportionand placed in the aerosol delivery system as described below. Further,as alluded to above, the particulate compositions (with or without anassociated agent) may be combined with one or more conventional (e.g. amicronized drug) active or bioactive agents to provide the desireddispersion stability or powder dispersibility.

Based on the foregoing, it will be appreciated by those skilled in theart that a wide variety of active agents may be incorporated in thedisclosed particulate compositions. Accordingly, the list of preferredactive agents above is exemplary only and not intended to be limiting.It will also be appreciated by those skilled in the art that the properamount of agent and the timing of the dosages may be determined for theparticulate compositions in accordance with already existing informationand without undue experimentation.

In addition to the phospholipid and polyvalent cation, themicroparticles of the present invention may also include abiocompatible, preferably biodegradable polymer, copolymer, or blend orother combination thereof. In this respect useful polymers comprisepolylactides, polylactide-glycolides, cyclodextrins, polyacrylates,methylcellulose, carboxymethylcellulose, polyvinyl alcohols,polyanhydrides, polylactams, polyvinyl pyrrolidones, polysaccharides(dextrans, starches, chitin, chitosan, etc.), hyaluronic acid, proteins,(albumin, collagen, gelatin, etc.). Examples of polymeric resins thatwould be useful for the preparation of perforated ink microparticlesinclude: styrene-butadiene, styrene-isoprene, styrene-acrylonitrile,ethylene-vinyl acetate, ethylene-acrylate, ethylene-acrylic acid,ethylene-methylacrylatate, ethylene-ethyl acrylate, vinyl-methylmethacrylate, acrylic acid-methyl methacrylate, and vinyl chloride-vinylacetate. Those skilled in the art will appreciate that, by selecting theappropriate polymers, the delivery efficiency of the particulatecompositions and/or the stability of the dispersions may be tailored tooptimize the effectiveness of the active or agent.

Besides the aforementioned polymer materials and surfactants, it may bedesirable to add other excipients to a particulate composition toimprove particle rigidity, production yield, emitted dose anddeposition, shelf-life and patient acceptance. Such optional excipientsinclude, but are not limited to: coloring agents, taste masking agents,buffers, hygroscopic agents, antioxidants, and chemical stabilizers.Further, various excipients may be incorporated in, or added to, theparticulate matrix to provide structure and form to the particulatecompositions (i.e. microspheres such as latex particles). In this regardit will be appreciated that the rigidifying components can be removedusing a post-production technique such as selective solvent extraction.

Other excipients may include, but are not limited to, carbohydratesincluding monosaccharides, disaccharides and polysaccharides. Forexample, monosaccharides such as dextrose (anhydrous and monohydrate),galactose, mannitol, D-mannose, sorbitol, sorbose and the like;disaccharides such as lactose, maltose, sucrose, trehalose, and thelike; trisaccharides such as raffinose and the like; and othercarbohydrates such as starches (hydroxyethylstarch), cyclodextrins andmaltodextrins. Other excipients suitable for use with the presentinvention, including amino acids, are known in the art such as thosedisclosed in WO 95/31479, WO 96/32096, and WO 96/32149. Mixtures ofcarbohydrates and amino acids are further held to be within the scope ofthe present invention. The inclusion of both inorganic (e.g. sodiumchloride, etc.), organic acids and their salts (e.g. carboxylic acidsand their salts such as sodium citrate, sodium ascorbate, magnesiumgluconate, sodium gluconate, tromethamine hydrochloride, etc.) andbuffers is also contemplated. The inclusion of salts and organic solidssuch as ammonium carbonate, ammonium acetate, ammonium chloride orcamphor are also contemplated.

Yet other preferred embodiments include particulate compositions thatmay comprise, or may be coated with, charged species that prolongresidence time at the point of contact or enhance penetration throughmucosae. For example, anionic charges are known to favor mucoadhesionwhile cationic charges may be used to associate the formedmicroparticulate with negatively charged bioactive agents such asgenetic material. The charges may be imparted through the association orincorporation of polyanionic or polycationic materials such aspolyacrylic acids, polylysine, polylactic acid and chitosan.

According to a preferred embodiment, the particulate compositions may beused in the form of dry powders or in the form of stabilized dispersionscomprising a non-aqueous phase. Accordingly, the dispersions or powdersof the present invention may be used in conjunction with metered doseinhalers (MDIs), dry powder inhalers (DPIs), atomizers, nebulizers orliquid dose instillation (LDI) techniques to provide for effective drugdelivery. With respect to inhalation therapies, those skilled in the artwill appreciate that the hollow and porous microparticles of the presentinvention are particularly useful in DPIs. Conventional DPIs comprisepowdered formulations and devices where a predetermined dose ofmedicament, either alone or in a blend with lactose carrier particles,is delivered as an aerosol of dry powder for inhalation.

The medicament is formulated in a way such that it readily dispersesinto discrete particles with an MMD between 0.5 to 20 μm, preferably0.5-5 μm, and are further characterized by an aerosol particle sizedistribution less than about 10 μm mass median aerodynamic diameter(MMAD), and preferably less than 5.0 μm. The mass median aerodynamicdiameters of the powders will characteristically range from about 0.5-10μm, preferably from about 0.5-5.0 μm MMAD, more preferably from about1.0-4.0 μm MMAD.

The powder is actuated either by inspiration or by some externaldelivery force, such as pressurized air. Examples of DPIs suitable foradministration of the particulate compositions of the present inventionare disclosed in U.S. Pat. Nos. 5,740,794, 5,785,049, 5,673,686, and4,995,385 and PCT application nos. 00/72904, 00/21594, and 01/00263,hereby incorporated in their entirety by reference. DPI formulations aretypically packaged in single dose units such as those disclosed in theabove mentioned patents or they employ reservoir systems capable ofmetering multiple doses with manual transfer of the dose to the device.

As discussed above, the stabilized dispersions disclosed herein may alsobe administered to the nasal or pulmonary air passages of a patient viaaerosolization, such as with a metered dose inhaler. The use of suchstabilized preparations provides for superior dose reproducibility andimproved lung deposition as disclosed in WO 99/16422, herebyincorporated in its entirety by reference. MDIs are well known in theart and could easily be employed for administration of the claimeddispersions without undue experimentation. Breath activated MDIs, aswell as those comprising other types of improvements which have been, orwill be, developed are also compatible with the stabilized dispersionsand present invention and, as such, are contemplated as being within thescope thereof. However, it should be emphasized that, in preferredembodiments, the stabilized dispersions may be administered with an MDIusing a number of different routes including, but not limited to,topical, nasal, pulmonary or oral. Those skilled in the art willappreciate that, such routes are well known and that the dosing andadministration procedures may be easily derived for the stabilizeddispersions of the present invention.

Along with the aforementioned embodiments, the stabilized dispersions ofthe present invention may also be used in conjunction with nebulizers asdisclosed in PCT WO 99/16420, the disclosure of which is herebyincorporated in its entirety by reference, in order to provide anaerosolized medicament that may be administered to the pulmonary airpassages of a patient in need thereof. Nebulizers are well known in theart and could easily be employed for administration of the claimeddispersions without undue experimentation. Breath activated nebulizers,as well as those comprising other types of improvements which have been,or will be, developed are also compatible with the stabilizeddispersions and present invention and are contemplated as being with inthe scope thereof.

Along with DPIs, MDIs and nebulizers, it will be appreciated that thestabilized dispersions of the present invention may be used inconjunction with liquid dose instillation or LDI techniques as disclosedin, for example, WO 99/16421 hereby incorporated in its entirety byreference. Liquid dose instillation involves the direct administrationof a stabilized dispersion to the lung. In this regard, direct pulmonaryadministration of bioactive compounds is particularly effective in thetreatment of disorders especially where poor vascular circulation ofdiseased portions of a lung reduces the effectiveness of intravenousdrug delivery. With respect to LDI the stabilized dispersions arepreferably used in conjunction with partial liquid ventilation or totalliquid ventilation. Moreover, the present invention may further compriseintroducing a therapeutically beneficial amount of a physiologicallyacceptable gas (such as nitric oxide or oxygen) into the pharmaceuticalmicrodispersion prior to, during or following administration.

Particularly preferred embodiments of the invention incorporate spraydried, hollow and porous particulate compositions as disclosed in WO99/16419, hereby incorporated in its entirety by reference. Suchparticulate compositions comprise particles having a relatively thinporous wall defining a large internal void, although, other voidcontaining or perforated structures are contemplated as well. Inpreferred embodiments the particulate compositions will further comprisean active agent.

Compositions according to the present invention typically yield powderswith bulk densities less than 0.5 g/cm³ or 0.3 g/cm³, preferably less0.1 g/cm³ and most preferably less than 0.05 g/cm³. By providingparticles with very low bulk density, the minimum powder mass that canbe filled into a unit dose container is reduced, which eliminates theneed for carrier particles. That is, the relatively low density of thepowders of the present invention provides for the reproducibleadministration of relatively low dose pharmaceutical compounds.Moreover, the elimination of carrier particles will potentially minimizethroat deposition and any “gag” effect, since the large lactoseparticles will impact the throat and upper airways due to their size.

It will be appreciated that the particulate compositions disclosedherein comprise a structural matrix that exhibits, defines or comprisesvoids, pores, defects, hollows, spaces, interstitial spaces, apertures,perforations or holes. The absolute shape (as opposed to the morphology)of the perforated microstructure is generally not critical and anyoverall configuration that provides the desired characteristics iscontemplated as being within the scope of the invention. Accordingly,preferred embodiments can comprise approximately microspherical shapes.However, collapsed, deformed or fractured particulates are alsocompatible.

In accordance with the teachings herein the particulate compositionswill preferably be provided in a “dry” state. That is the microparticleswill possess a moisture content that allows the powder to remainchemically and physically stable during storage at ambient temperatureand easily dispersible. As such, the moisture content of themicroparticles is typically less than 6% by weight, and preferably less3% by weight. In some instances the moisture content will be as low as1% by weight. Of course it will be appreciated that the moisture contentis, at least in part, dictated by the formulation and is controlled bythe process conditions employed, e.g., inlet temperature, feedconcentration, pump rate, and blowing agent type, concentration and postdrying.

Reduction in bound water leads to significant improvements in thedispersibility and flowability of phospholipid based powders, leading tothe potential for highly efficient delivery of powdered lung surfactantsor particulate composition comprising active agent dispersed in thephospholipid. The improved dispersibility allows simple passive DPIdevices to be used to effectively deliver these powders.

Although the powder compositions are preferably used for inhalationtherapies, the powders of the present invention can also be administeredby other techniques known in the art, including, but not limited tointramuscular, intravenous, intratracheal, intraperitoneal,subcutaneous, and transdermal, either as dry powders, reconstitutedpowders, or suspensions.

As seen from the passages above, various components may be associatedwith, or incorporated in the particulate compositions of the presentinvention. Similarly, several techniques may be used to provideparticulates having the desired morphology (e.g. a perforated orhollow/porous configuration), dispersibility and density. Among othermethods, particulate compositions compatible with the instant inventionmay be formed by techniques including spray drying, vacuum drying,solvent extraction, emulsification or lyophilization, and combinationsthereof. It will further be appreciated that the basic concepts of manyof these techniques are well known in the prior art and would not, inview of the teachings herein, require undue experimentation to adaptthem so as to provide the desired particulate compositions.

While several procedures are generally compatible with the presentinvention, particularly preferred embodiments typically compriseparticulate compositions formed by spray drying. As is well known, spraydrying is a one-step process that converts a liquid feed to a driedparticulate form. With respect to pharmaceutical applications, it willbe appreciated that spray drying has been used to provide powderedmaterial for various administrative routes including inhalation. See,for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols:Physical and Biological Basis for Therapy, A. J. Hickey, ed. MarcelDekkar, New York, 1996, which is incorporated herein by reference.

In general, spray drying consists of bringing together a highlydispersed liquid, and a sufficient volume of hot air to produceevaporation and drying of the liquid droplets. The preparation to bespray dried or feed (or feed stock) can be any solution, coursesuspension, slurry, colloidal dispersion, or paste that may be atomizedusing the selected spray drying apparatus. In preferred embodiments thefeed stock will comprise a colloidal system such as an emulsion, reverseemulsion, microemulsion, multiple emulsion, particulate dispersion, orslurry. Typically the feed is sprayed into a current of warm filteredair that evaporates the solvent and conveys the dried product to acollector. The spent air is then exhausted with the solvent. Thoseskilled in the art will appreciate that several different types ofapparatus may be used to provide the desired product. For example,commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. willeffectively produce particles of desired size.

It will further be appreciated that these spray dryers, and specificallytheir atomizers, may be modified or customized for specializedapplications, i.e. the simultaneous spraying of two solutions using adouble nozzle technique. More specifically, a water-in-oil emulsion canbe atomized from one nozzle and a solution containing an anti-adherentsuch as mannitol can be co-atomized from a second nozzle. In other casesit may be desirable to push the feed solution though a custom designednozzle using a high pressure liquid chromatography (HPLC) pump. Providedthat microstructures comprising the correct morphology and/orcomposition are produced the choice of apparatus is not critical andwould be apparent to the skilled artisan in view of the teachingsherein. Examples of spray drying methods and systems suitable for makingthe dry powders of the present invention are disclosed in U.S. Pat. Nos.6,077,543, 6,051,256, 6,001,336, 5,985,248, and 5,976,574, herebyincorporated in their entirety by reference.

While the resulting spray-dried powdered particles typically areapproximately spherical in shape, nearly uniform in size and frequentlyare hollow, there may be some degree of irregularity in shape dependingupon the incorporated medicament and the spray drying conditions. Inmany instances dispersion stability and dispersibility of theparticulate compositions appears to be improved if an inflating agent(or blowing agent) is used in their production as disclosed in WO99/16419 cited above. Particularly preferred embodiments comprise anemulsion with the inflating agent as the disperse or continuous phase.The inflating agent is preferably dispersed with a surfactant solution,using, for instance, a commercially available microfluidizer at apressure of about 5000 to 15,000 psi. This process forms an emulsion,preferably stabilized by an incorporated surfactant, typicallycomprising submicron droplets of water immiscible blowing agentdispersed in an aqueous continuous phase. The formation of suchemulsions using this and other techniques are common and well known tothose in the art. The blowing agent is preferably a fluorinated compound(e.g. perfluorohexane, perfluorooctyl bromide, perfluorooctyl ethane,perfluorodecalin, perfluorobutyl ethane) which vaporizes during thespray-drying process, leaving behind generally hollow, porousaerodynamically light microspheres. Other suitable liquid blowing agentsinclude nonfluorinated oils, chloroform, Freons, ethyl acetate, alcoholsand hydrocarbons. Nitrogen and carbon dioxide gases are alsocontemplated as a suitable blowing agent. Perfluorooctyl ethane isparticularly preferred according to the invention.

Besides the aforementioned compounds, inorganic and organic substanceswhich can be removed under reduced pressure by sublimation in apost-production step are also compatible with the instant invention.These sublimating compounds can be dissolved or dispersed as micronizedcrystals in the spray drying feed solution and include ammoniumcarbonate and camphor. Other compounds compatible with the presentinvention comprise rigidifying solid structures which can be dispersedin the feed solution or prepared in-situ. These structures are thenextracted after the initial particle generation using a post-productionsolvent extraction step. For example, latex particles can be dispersedand subsequently dried with other wall forming compounds, followed byextraction with a suitable solvent.

Although the particulate compositions are preferably formed using ablowing agent as described above, it will be appreciated that, in someinstances, no additional blowing agent is required and an aqueousdispersion of the medicament and/or excipients and surfactant(s) arespray dried directly. In such cases, the formulation may be amenable toprocess conditions (e.g., elevated temperatures) that may lead to theformation of hollow, relatively porous microparticles. Moreover, themedicament may possess special physicochemical properties (e.g., highcrystallinity, elevated melting temperature, surface activity, etc.)that makes it particularly suitable for use in such techniques.

Regardless of which blowing agent is ultimately selected, it has beenfound that compatible particulate compositions may be producedparticularly efficiently using a Büchi mini spray drier (model B-191,Switzerland). As will be appreciated by those skilled in the art, theinlet temperature and the outlet temperature of the spray drier are notcritical but will be of such a level to provide the desired particlesize and to result in a product that has the desired activity of themedicament. In this regard, the inlet and outlet temperatures areadjusted depending on the melting characteristics of the formulationcomponents and the composition of the feed stock. The inlet temperaturemay thus be between 60° C. and 170° C., with the outlet temperatures ofabout 40° C. to 120° C. depending on the composition of the feed and thedesired particulate characteristics. Preferably these temperatures willbe from 90° C. to 120° C. for the inlet and from 60° C. to 90° C. forthe outlet. The flow rate which is used in the spray drying equipmentwill generally be about 3 ml per minute to about 15 ml per minute. Theatomizer air flow rate will vary between values of 25 liters per minuteto about 50 liters per minute. Commercially available spray dryers arewell known to those in the art, and suitable settings for any particulardispersion can be readily determined through standard empirical testing,with due reference to the examples that follow. Of course, theconditions may be adjusted so as to preserve biological activity inlarger molecules such as proteins or peptides.

Whatever components are selected, the first step in particulateproduction typically comprises feed stock preparation. If thephospholipid based particle is intended to act as a carrier for anotheractive agent, the selected active agent is dissolved in a solvent,preferably water, to produce a concentrated solution. The polyvalentcation may be added to the active agent solution or may be added to thephospholipid emulsion as discussed below. The active agent may also bedispersed directly in the emulsion, particularly in the case of waterinsoluble agents. Alternatively, the active agent may be incorporated inthe form of a solid particulate dispersion. The concentration of theactive agent used is dependent on the amount of agent required in thefinal powder and the performance of the delivery device employed (e.g.,the fine particle dose for a MDI or DPI). As needed, cosurfactants suchas poloxamer 188 or span 80 may be dispersed into this annex solution.Additionally, excipients such as sugars and starches can also be added.

In selected embodiments a polyvalent cation-containing oil-in-wateremulsion is then formed in a separate vessel. The oil employed ispreferably a fluorocarbon (e.g., perfluorooctyl bromide, perfluorooctylethane, perfluorodecalin) which is emulsified with a phospholipid. Forexample, polyvalent cation and phospholipid may be homogenized in hotdistilled water (e.g., 60° C.) using a suitable high shear mechanicalmixer (e.g., Ultra-Turrax model T-25 mixer) at 8000 rpm for 2 to 5minutes. Typically 5 to 25 g of fluorocarbon is added dropwise to thedispersed surfactant solution while mixing. The resulting polyvalentcation-containing perfluorocarbon in water emulsion is then processedusing a high pressure homogenizer to reduce the particle size. Typicallythe emulsion is processed at 12,000 to 18,000 psi, 5 discrete passes andkept at 50 to 80° C.

The active agent solution and perfluorocarbon emulsion are then combinedand fed into the spray dryer. Typically the two preparations will bemiscible as the emulsion will preferably comprise an aqueous continuousphase. While the bioactive agent is solubilized separately for thepurposes of the instant discussion it will be appreciated that, in otherembodiments, the active agent may be solubilized (or dispersed) directlyin the emulsion. In such cases, the active emulsion is simply spraydried without combining a separate active agent preparation.

In any event, operating conditions such as inlet and outlet temperature,feed rate, atomization pressure, flow rate of the drying air, and nozzleconfiguration can be adjusted in accordance with the manufacturer'sguidelines in order to produce the required particle size, andproduction yield of the resulting dry particles. Exemplary settings areas follows: an air inlet temperature between 60° C. and 170° C.; an airoutlet between 40° C. to 120° C.; a feed rate between 3 ml to about 15ml per minute; and an aspiration air flow of 300 L/min. and anatomization air flow rate between 25 to 50 L/min. The selection ofappropriate apparatus and processing conditions are well within thepurview of a skilled artisan in view of the teachings herein and may beaccomplished without undue experimentation. In any event, the use ofthese and substantially equivalent methods provide for the formation ofhollow porous aerodynamically light microparticles with particlediameters appropriate for aerosol deposition into the lung.microstructures that are both hollow and porous, almost honeycombed orfoam-like in appearance. In especially preferred embodiments theparticulate compositions comprise hollow, porous spray driedmicroparticles.

Along with spray drying, particulate compositions useful in the presentinvention may be formed by lyophilization. Those skilled in the art willappreciate that lyophilization is a freeze-drying process in which wateris sublimed from the composition after it is frozen. The particularadvantage associated with the lyophilization process is that biologicalsand pharmaceuticals that are relatively unstable in an aqueous solutioncan be dried without elevated temperatures (thereby eliminating theadverse thermal effects), and then stored in a dry state where there arefew stability problems. With respect to the instant invention suchtechniques are particularly compatible with the incorporation ofpeptides, proteins, genetic material and other natural and syntheticmacromolecules in particulate compositions without compromisingphysiological activity. Methods for providing lyophilized particulatesare known to those of skill in the art and it would clearly not requireundue experimentation to provide dispersion compatible microparticles inaccordance with the teachings herein. The lyophilized cake containing afine foam-like structure can be micronized using techniques known in theart to provide 3 to 10 μm sized particles. Accordingly, to the extentthat lyophilization processes may be used to provide microparticleshaving the desired porosity and size they are in conformance with theteachings herein and are expressly contemplated as being within thescope of the instant invention.

Besides the aforementioned techniques, the particulate compositions orparticles of the present invention may also be formed using a methodwhere a feed solution (either emulsion or aqueous) containing wallforming agents is rapidly added to a reservoir of heated oil (e.g.perflubron or other high boiling FCs) under reduced pressure. The waterand volatile solvents of the feed solution rapidly boils and areevaporated. This process provides a perforated structure from the wallforming agents similar to puffed rice or popcorn. Preferably the wallforming agents are insoluble in the heated oil. The resulting particlescan then separated from the heated oil using a filtering technique andsubsequently dried under vacuum.

Additionally, the particulate compositions of the present invention mayalso be formed using a double emulsion method. In the double emulsionmethod the medicament is first dispersed in a polymer dissolved in anorganic solvent (e.g. methylene chloride, ethyl acetate) by sonicationor homogenization. This primary emulsion is then stabilized by forming amultiple emulsion in a continuous aqueous phase containing an emulsifiersuch as polyvinylalcohol. Evaporation or extraction using conventionaltechniques and apparatus then removes the organic solvent. The resultingmicrospheres are washed, filtered and dried prior to combining them withan appropriate suspension medium in accordance with the presentinvention

Whatever production method is ultimately selected for production of theparticulate compositions, the resulting powders have a number ofadvantageous properties that make them particularly compatible for usein devices for inhalation therapies. In particular, the physicalcharacteristics of the particulate compositions make them extremelyeffective for use in dry powder inhalers and in the formation ofstabilized dispersions that may be used in conjunction with metered doseinhalers, nebulizers and liquid dose instillation. As such, theparticulate compositions provide for the effective pulmonaryadministration of active agents.

In order to maximize dispersibility, dispersion stability and optimizedistribution upon administration, the mean geometric particle size ofthe particulate compositions is preferably about 0.5-50 μm, morepreferably 1-20 μm and most preferably 0.5-5 μm. It will be appreciatedthat large particles (i.e. greater than 50 μm) may not be preferred inapplications where a valve or small orifice is employed, since largeparticles tend to aggregate or separate from a suspension which couldpotentially clog the device. In especially preferred embodiments themean geometric particle size (or diameter) of the particulatecompositions is less than 20 μm or less than 10 μm. More preferably themean geometric diameter is less than about 7 μm or 5 μm, and even morepreferably less than about 2.5 μm. Other preferred embodiments willcomprise preparations wherein the mean geometric diameter of theparticulate compositions is between about 1 μm and 5 μm. In especiallypreferred embodiments the particulate compositions will comprise apowder of dry, hollow, porous microspherical shells of approximately 1to 10 μm or 1 to 5 μm in diameter, with shell thicknesses ofapproximately 0.1 μm to approximately 0.5 μm. It is a particularadvantage of the present invention that the particulate concentration ofthe dispersions and structural matrix components can be adjusted tooptimize the delivery characteristics of the selected particle size.

Although preferred embodiments of the present invention comprise powdersand stabilized dispersions for use in pharmaceutical applications, itwill be appreciated that the particulate compositions and discloseddispersions may be used for a number of non pharmaceutical applications.That is, the present invention provides particulate compositions whichhave a broad range of applications where a powder is suspended and/oraerosolized. In particular, the present invention is especiallyeffective where an active or bioactive ingredient must be dissolved,suspended or solubilized as fast as possible. By increasing the surfacearea of the porous microparticles or by incorporation with suitableexcipients as described herein, will result in an improvement indispersibility, and/or suspension stability. In this regard, rapiddispersement applications include, but are not limited to: detergents,dishwasher detergents, food sweeteners, condiments, spices, mineralflotation detergents, thickening agents, foliar fertilizers,phytohormones, insect pheromones, insect repellents, pet repellents,pesticides, fungicides, disinfectants, perfumes, deodorants, etc.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, merelyrepresentative of preferred methods of practicing the present inventionand should not be read as limiting the scope of the invention.

Example I Effect of Added Calcium Ions on the Tm of Spray-DriedPhospholipids

The effect of calcium ions on the gel-to-liquid crystal transitiontemperature (Tm) of spray-dried phospholipids was investigated. Theresulting powders were examined visually for powder flowcharacteristics, characterized for Tm using a differential scanningcalorimeter (DSC).

Dry lung surfactant particles comprising long-chain saturatedphosphatidylcholines, PCs (e.g., dipalmitoylphosphatidylcholine, DPPC ordistearoylphosphatidylcholine, DSPC) and varying amounts of calciumchloride were manufactured by an emulsion-based spray-drying process.Calcium levels were adjusted as mole ratio equivalents relative to thePC present, with Ca/PC (mol/mol)=0 to 1. Accordingly, 1 g of saturatedphosphatidylcholine (Genzyme Corp, Cambridge, Mass.) and 0 to 0.18 g ofcalcium chloride dihydrate (Fisher Scientific Corp., Pittsburgh, Pa.)were dispersed in approximately 40 mL of hot deionized water (T=60-70°C.) using an Ultra-Turrax T-25 mixer at 8,000-10,000 rpm for 2 to 5minutes. 18 g of perfluorooctyl ethane, PFOE (F-Tech, Tokyo, Japan) wasthen added dropwise during mixing at a rate of 2-5 ml/min. After theaddition was complete, the emulsion was mixed for an additional periodof not less than 4 minutes at 10,000-12,000 rpm. The resulting coarseemulsion was then homogenized under high pressure with an Avestin C-5homogenizer (Ottawa, Canada) at 8,000-10,000 psi for 4 passes, and at18,000-20,000 psi for a final pass.

The submicron fluorocarbon-in-water emulsion was then spray-dried with aBuchi B-191 Mini Spray-Drier (Flawil, Switzerland), equipped with amodified 2-fluid atomizer under the following conditions: inlettemperature=85° C.; outlet temperature=58°-61° C.; pump=1.9 ml min⁻¹;atomizer pressure=60-65 psig; atomizer flow rate=30-35 cm. Theaspiration flow (69-75%) was adjusted to maintain an exhaust bagpressure=20-21 mbar.

The spray-dried phospholipid particles were collected using the standardBuchi cyclone separator. The volume-weighted mean geometric diameter(VMD) of the dry phospholipid particles was confirmed by laserdiffraction (Sympatech Helos H1006, Clausthal-Zellerfeld, Germany), andranged from 2.5 μm to 3.8 μm depending on the formulation.

The resulting dry phospholipid particles were also characterized using amodel 2920 DSC (TA Instruments) and by a Karl Fisher moisture analyzer.Approximately 0.5 to 2 mg dry powder was weighed into aluminum samplepans and hermetically sealed. Each sample was analyzed using a modulatedDSC mode under the following conditions: equilibration at −20° C., and2° C./min ramp to 150° C. modulated +/−1° C. every 60 sec. Thephospholipid Tm was defined as the peak maxima of the first endothermictransition from each reversing heat flow thermogram. For moistureanalysis, approximately 50 mg powder was suspended in 1 mL of anhydrousdimethylforamide (DMF). The suspension was then injected directly intothe titration cell and the moisture content was derived. The residualmoisture content in the spray-dried DSPC particles is shown in Table Ia,and was found to decrease as a function of Ca/PC mole ratio. Tables Iband Ic present the Tm values for the various spray-dried PC particles asa function of the Ca/PC ratio. Hydrated DSPC and DPPC liposomes exhibitTm values of 58 and 42° C., respectively. Dramatic increases in Tm wereobserved following spray-drying, and with increases in calcium content.The powder formulations devoid of calcium ions were highly cohesive,while the formulations incorporating added calcium were free-flowingpowders.

The present example illustrates that the hydration status of powderedphospholipid preparations greatly influences their inherentthermodynamic and physicochemical characteristics, i.e., Tm and flowproperties. Increases in phospholipid Tm are believed to directlycorrelate with increases in thermal stability, which could lead to anenhancement in long-term storage stability. In addition, decreasedmoisture content may also lead to greater chemical stability.

TABLE Ia Effect of Added Calcium on the Residual Moisture Content ofSpray-Dried DSPC Ca/DSPC (mol/mol) Water Content (%) 0 2.9 0.25 1.9 0.501.4

TABLE Ib Effect of Added Calcium on the Tm of Spray-dried DSPC Ca/DSPC(mol/mol) Tm (° C.) 0 (hydrated) 58 0 79 0.25 85 0.5 98 1.0 126

TABLE Ic Effect of Added Calcium on the Tm of Spray-dried DPPC Ca/DPPC(mol/mol) Tm (° C.) 0 (hydrated) 42 0 63 0.25 69 0.5 89

Example II Effect of Added Magnesium Ions on Tm of Spray-DriedPhospholipids

Phospholipid particles stabilized with magnesium ions were prepared byan emulsion-based spray-drying technique. The emulsion feedstock wasprepared according to the procedure described below. In the first step,0.45 g of distearoylphosphatidylcholine, DSPC, and 0.126 g magnesiumchloride hexahydrate (Fisher Scientific, Pittsburgh, Pa.) were dispersedin 41 g of hot deionized water (T=60 to 70° C.) using an Ultra-Turraxmixer (model T-25) at 10,000 rpm for 2 min. 17 g of perfluorooctylethane was then added drop wise at a rate of approximately 1-2 ml/minduring mixing. After the fluorocarbon addition was complete, theemulsion was mixed for an additional period of not less than 4 minutes.The resulting coarse emulsion was then processed through a high pressurehomogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes, toyield a submicron fluorocarbon-in-water emulsion stabilized by amonolayer of DSPC. The emulsion was then spray-dried with a Buchi modelB-191 Mini Spray-Drier under the following spray conditions:aspiration=69%, inlet temperature=85° C., outlet temperature=58° C.,feed pump=1.9 mL min⁻¹, and atomizer flow rate=33 cm.

Differential scanning calorimetric analysis of the dry particlesrevealed the Tm for the DSPC in the powder was 88° C. as compared with79° C. for neat DSPC (Table Ib). This foregoing example illustrates theeffect ions such as magnesium have upon the thermodynamic properties ofdry phospholipid particles.

Example III Preparation of Spray-Dried Lung Surfactant (ExoSurf®)Particles

Dry lung surfactant particles having the same components as ExoSurf(Glaxo-Wellcome, Research Triangle Park, N.C.) were manufactured using aspray-drying process. To achieve this end, the osmotic NaCl component ofExosurf was replaced in one formulation by CaCl₂. Accordingly, 1.55 g ofdipalmitoylphosphatidylcholine and 0.144 g of calcium chloride dihydrateor sodium chloride were dispersed in 50 mL of hot deionized water(T=60-70° C.) using an Ultra-Turrax T-25 mixer at 8,000-10,000 rpm for 2min. 18.5 g of perfluorooctyl ethane was then added dropwise duringmixing at a rate of 2-5 ml/min. After the addition was complete, theemulsion was mixed for an additional period of not less than 4 minutesat 10,000-12,000 rpm. The resulting coarse emulsion was then homogenizedunder high pressure with an Avestin C-5 homogenizer (Ottawa, Canada) at8,000-10,000 psi for 4 passes, and at 18,000-20,000 psi for a finalpass. In a separate flask, 0.12 g of Tyloxapol® was dispersed in 10 g ofhot deionized water (T=60-70° C.). The Tyloxapol dispersion was thendecanted into a vial that contained 0.174 g of cetyl alcohol. The vialwas sealed and the cetyl alcohol was dispersed by placing it in asonication bath for 15 minutes. The Tyloxapol/cetyl alcohol dispersionwas added to the fluorocarbon emulsion and mixed for 5 min. The feedsolution was then spray-dried with a Bucchi-191 Mini Spray-Drier,equipped with a modified 2-fluid atomizer under the followingconditions: inlet temperature=85° C., outlet temperature=58°-61° C.,pump=1.9 ml min⁻¹, atomizer pressure=60-65 psig, atomizer flowrate=30-35 cm. The aspiration flow (69-75%) was adjusted to maintain anexhaust bag pressure=20-21 mbar. A free flowing white powder wascollected using the standard Buchi cyclone separator.

The spray-dried powders were manually filled into a proprietary blisterpackage and heat-sealed. The filling procedure was performed in ahumidity controlled glove box (RH<2%). All blister packages werenumbered, then weighed before and after filling to determine the amountof powder loaded. The filled blister packages were stored in adesiccating box operated at <2% RH until use. The powders were thentested for dispersibility from a DPI described in U.S. Pat. No.5,740,794.

Emitted Dose testing of the formulations was assessed following USPguidelines for inhalation products. The actuated dose was collectedusing a 30 L min⁻¹ flow rate held for 2 seconds onto a type A/E glassfilter (Gelman, Ann Arbor, Mich.). The emitted dose was calculatedgravimetrically knowing the blister weight, total blister fill weight,and net change in filter weight.

Dry powder containing sodium chloride exhibited poor powder flow, anddid not aerosolize well. In contrast, the formulation in which calciumchloride was substituted for the sodium chloride yielded particles withgood flow and excellent emitted dose character. The differences indispersibility between the two formulations is further reflected in thestandard deviations of the emitted dose. The foregoing exampleillustrates the ability of the present invention to alter and modulatethe flow and emission properties of dry lipid particles through theinclusion of calcium ions.

TABLE II Formulation of Highly Dispersible Dry Powder Lung SurfactantPreparations Dry Powder Ca/DSPC Emitted Dose Formulation (mol/mol) (%)“Exosurf” 0 10 ± 33 “Exosurf” + Calcium 0.5 87 ± 3 

Example IV Thermal Stability of Spray-Dried Phospholipid Particles

In the current example, the thermal stability of the spray-driedphospholipid particles prepared in example I were assessed. Accordingly50 mg of powder was transferred into 20 mL glass vials and stored in avacuum oven at 100° C. for 1 hour. The volume-weighted mass mediandiameters (MMD) for the powders were determined using a SympaTech laserdiffraction analyzer (HELOS H1006, Clausthal-Zellerfeld, Germany)equipped with a RODOS type T4.1 vibrating trough. Approximately 1-3 mgof powder was placed in the powder feeder, which was subsequentlyatomized through a laser beam using 1 bar of air pressure, 60 mbar ofvacuum, 70% feed rate and 1.30 mm funnel gap. Data was collected over aninterval of 0.4 s, with a 175 μm focal length, triggered at 1%obscuration. Particle size distributions were determined using aFraünhofer model. The volume-weighted mean aerodynamic diameters (VMAD)for the powders were determined with a model 8050 Aerosizer®LD particlesize analysis system (Amherst Process Instruments, Hadley, Mass.)equipped with an Aero-Sampler® chamber. Approximately 0.2 mg of powderwas loaded into a specially designed DPI testing apparatus. In thistest, the powder was aerosolized by actuating a propellant cancontaining HFA-134a through the loaded sample chamber. The design ofthis apparatus is such to mimic actuation from an active DPI device andto offer some insight into powder flowability or its ability todeaggregate. Narrow particle size distributions are preferred and arebelieved to be an indication of the powder's ability to deaggregate.

Table III depicts the thermal stability and changes in particle size(MMD and VMAD) for the various spray-dried DSPC particles as a functionof Ca/DSPC (mol/mol) ratio. The thermal stability of the powders wasfound to increase with increasing calcium content. Significantstructural and particle size changes were observed for the formulationdevoid of calcium ions, as evidenced by particle sintering and largeincreases in MMD and VMAD. The addition of small amounts of calcium ions(Ca/DSPC=0.25) resulting in a significant improvement in thermalstability of the phospholipid particles. More surprising, thespray-dried phospholipid formulation enriched at Ca/DSPC ratio of 0.5completely tolerated the accelerated storage conditions, as nosignificant changes had occurred as a result of storage at 100° C. for 1hour. The above example further illustrates the enhanced thermalstability of spray-dried phospholipid particles afforded by theinclusion of calcium ions.

TABLE III Aerosol characteristics of Spray-Dried DSPC Powders followingStorage at 100° C. for 1 hour Ca/ DSPC Tm Thermal MMD₀ VMAD₀ MMD VMAD(mol/mol) (° C.) Stability (μm) (μm) (μm) (μm) 0 79 Sintering 3.3 2.15.7 4.1 at 5 min. 0.25 85 Sintering 3.4 1.8 4.5 2.1 at 45 min 0.5 98 NoChange 3.6 1.7 3.5 1.8

Example V The Effect of Added Calcium Ions on pMDI Stability

The objective of this study was to examine the effect added calcium hadon the physical stability of lipid-based pMDI suspensions to moisture.Budesonide powders were prepared by spray-drying a feed solutioncomprised of micronized drug particles suspended in the aqueous phase ofa fluorocarbon-in-water emulsion. Accordingly, 0.8 g saturated eggphosphatidylcholine (EPC-3, Lipoid KG, Ludwigshafen, Germany) wasdispersed in approximately 80 mL hot deionized water (T=80° C.) using anUltra-Turrax mixer at 8000 rpm for 2 to 5 minutes. 20 g of perflubron(φ=0.09) was then added drop wise during mixing. After the addition wascomplete, the emulsion was mixed for an additional period of not lessthan 4 minutes. The resulting coarse emulsion was homogenized under highpressure with an Avestin C-5 homogenizer (Ottawa, Canada) at 18,000 psifor 5 passes. The resulting submicron emulsion was then combined with asecond aqueous phase containing 1.33 g budesonide suspended in asolution comprising 0.4 g d-lactose monohydrate, and 0-0.134 g calciumchloride dissolved in approximately 30 g of deionized water. Thecombined solution was then mixed using an Ultra-Turrax mixer at 8000 rpmfor 2 minutes to ensure dispersion of the budesonide particles. Hollowporous budesonide particles were prepared by spray-drying the dispersionwith a B-191 Mini Spray-Drier (Büchi, Flawil, Switzerland) under thefollowing spray conditions: aspiration=80%, inlet temperature=85° C.,outlet temperature=57° C., feed pump=2.3 mL/min, total air flow=22.4SCFM. Free flowing white powders were collected at the cycloneseparator. Scanning electron microscopic (SEM) analysis showed thepowders to be spherical and highly porous.

Approximately 40 mg of spray-dried budesonide particles were weighedinto 10 ml aluminum cans, and crimp sealed (Pamasol 2005/10, Pfaffikon,Switzerland) with a DF30/50 ACT 50 μl metering valve (Valois of America,Greenwich, Conn.). The canisters were charged with 5 g HFA-134a (DuPont,Wilmington, Del.) propellant by overpressure through the valve stem(Pamasol 8808). To elucidate differences between the budesonideformulations, propellant preparations that were spiked with varyingamounts of water (0 to 1100 ppm) were utilized. The amount of thepropellant in the can was determined by weighing the can before afterthe fill. The final powder concentration in propellant was ˜0.8% w/w andformulated to provide a theoretical ex-valve dose of 100 μg budesonideper actuation. Powder dispersion was achieved by placing the canistersin a sonication bath for 15 min. The charged pMDIs were placed inquarantine for a period of 7 days at ambient conditions to allow thevalve seals to seat.

For the purpose of this study the aerosol fine particle fraction, FPF(%<5.8 μm) was used to assess changes in suspension physical stabilitythat had occurred as a result of the water activity. The budesonidepMDIs were tested using commonly accepted pharmaceutical procedures. Themethod utilized was compliant with the United State Pharmacopeia (USP)procedure (Pharmacopeial Previews (1996) 22:3065-3098). After 5 wasteshots, 20 doses from the test pMDIs were actuated into an AndersenImpactor. The extraction from all the plates, induction port, andactuator were performed in closed containers with an appropriate amountof methanol:water (1:1, v/v). The filter was installed but not assayed,because the polyacrylic binder interfered with the analysis. Budesonidewas quantified by measuring the absorption at 245 nm (Beckman DU640spectrophotometer) and compared to an external standard curve with theextraction solvent as the blank. The FPF was calculated according to theUSP method referenced above.

The effect of added calcium ions on the physical stability of thebudesonide pMDIs is depicted in FIG. 1. The physical stability of thebudesonide pMDIs was found to increase with increasing calciumconcentration. Surprisingly the tolerance of the budesonide pMDIsuspension to moisture increased from approximately 400 ppm to nearly700 ppm by the inclusion of 4% calcium chloride into the formulation.

This example illustrates the enhanced stability of phospholipid-basedpMDI particles afforded by the presence of calcium ions. The ability ofa pMDI formulation to tolerate increased levels of moisture will lead toan enhancement in their long-term storage stability. The presence ofwater fuels structural changes, which can lead to formation of liquidbridges between particles and/or recrystallization of components andchanges in surface characteristics. The overall effect of moistureingress for suspension pMDIs leads to particle coarsening and suspensioninstability, all of which can lead to product failure.

Example VI The Effect of Added Calcium Ions on Particle Morphology

The objective of this study was to examine the effect added calcium hasupon the morphological character of spray-dried phospholipid particles.Scanning electron micrographic (SEM) images of the spray-drieddistearoylphosphatidylcholine particles prepared in example I weretaken. The powders were placed on double sticky carbon graphite that wasaffixed on labeled aluminum stubs. The samples were then sputter-coatedwith a 250-300 Å layer of gold/palladium. Samples were examined on ascanning electron microscope operated at an accelerating voltage of 20Kev, and a probe current of 250 pAmps. Photomicrographs were digitallycaptured at a 20,000× magnification.

The effect of calcium ion concentration on the morphology of spray-driedDSPC particles is illustrated in Figure. II. Formulations containingcalcium ions had a highly porous sponge-like inflated morphology,whereas the neat DSPC particles appeared melted and collapsed. Thehollow porous morphology is characterized by powders that flow andaerosolize well, whereas the collapsed morphology results in powderswith poor flowability and dispersibility. No significant difference inmorphology was observed as a result of calcium ion concentration,although the Ca/DSPC=0.25 formulation exhibited some degree of meltedcharacter as well. The decreased sensitivity of the powders with highercalcium content to melting and particle fusion is likely the result ofthe increased Tm values that allow for the powders to experience ahigher drying temperature while maintaining the lipids in the gel state.The significant increases in Tm observed (Example I) lead to greaterflexibility in spray-drying manufacture of these particles, and asignificantly greater likelihood of achieving desired particlemorphologies which are dependent on drying rates.

Example VII Preparation of Spray-Dried Budesonide Particles

Hollow porous budesonide particles were prepared by a two-step process.In the first step, 54 mg of budesonide (Vinchem, Chatham, N.J.), and0.775 g of DSPC were dissolved in 2 ml of chloroform:methanol (2:1). Thechloroform:methanol was then evaporated to obtain a thin film of thephospholipid/steroid mixture. The phospholipid/steroid mixture was thendispersed in 30.5 g of hot deionized water (T=60 to 70° C.) using anUltra-Turrax mixer (model T-25) at 8000 rpm for 2 to 5 minutes. 12.8 gof perfluorooctyl ethane was then added dropwise during mixing. Afterthe addition was complete, the emulsion was mixed for an additionalperiod of not less than 4 minutes. The coarse emulsion was then passedthrough a high pressure homogenizer (Avestin, Ottawa, Canada) at 18,000psi for 5 passes. The resulting submicron fluorocarbon-in-water withsteroid solubilized in the lipid monolayer surrounding the droplets wasutilized as the feedstock in for the second step, i.e. spray-drying on aB-191 Mini Spray-Drier (Büchi, Flawil, Switzerland). Calcium chloride (0or 0.65 mg) was added in 2.5 g of water to the fluorocarbon-in-wateremulsion immediately prior to spray drying. The following sprayconditions were employed: aspiration=100%, inlet temperature=85° C.,outlet temperature=60° C., feed pump=1.9 mL min⁻¹, atomizerpressure=60-65 psig, atomizer flow rate=30-35 cm. The aspiration flow(69-75%) was adjusted to maintain an exhaust bag pressure of 30-31 mbar.Free flowing white powders were collected using a standard cycloneseparator.

The resulting dry budesonide particles were characterized using DSC.Each sample was analyzed in a modulated DSC mode under the followingconditions: equilibration at −20° C., and 2° C./min ramp to 150° C.modulated +/−1° C. every 60 sec. The phospholipid Tm was defined as thepeak maxima of the first endothermic transition from each reversing heatflow thermogram. The phospholipid Tm for DSPC particles without addedcalcium is 79° C. The addition of calcium ions in the budesonideformulation increased the Tm to 98° C. In addition, the powderformulations devoid of calcium had a cohesive flow character as comparedto the calcium-enriched formulation.

The aerosol characteristics of the calcium containing formulation wasexamined in several passive dry powder inhaler devices (Eclipse®,Turbospin®, Cipla Rotahaler®, Glaxo Rotahaler®®, and Hovione FlowCaps®).The emitted dose was determined gravimetrically at comfortableinhalation flow rate (peak flow rate=20-62 L/min depending on theresistance of the device), and at a forced inhalation flow rate (peakflow rate 37-90 L/min). Under comfortable inhalation flow conditions therange of emitted doses was between 89 and 96% with a mean emitted doseof 94%. Under forced inhalation flow, the emitted dose varied between 94and 103%, with a mean emitted dose of 99%. The fact that multipledevices with high and low resistance are able to effectively dispersethe powders more or less independent of inspiratory flow rate speaksvolumes to the dispersibility of the calcium containing budesonidepowder tested.

The above example further illustrates the ability of the present powderengineering technology to effectively modulate the Tm throughformulation changes. Increased (Tm's) are desired as they often indicateincreased physical stability and improved powder dispersibility.

Example VIII Rapid Spreading of Spray-Dried DSPC Particles on anAir-Water Interface

The rapid spreading characteristics of the disclosed spray-driedphospholipid-based particles at the air/water interface are illustratedin FIG. III. Surface tension measurements were made on a Kruss K12tensiometer at 25° C. using the Wilhemey plate technique. To measuresurface tension, 20 mL of DI water or DSPC liposome dispersion wasplaced in the thermostatic beaker. The platinum plate was tared in theair and then dipped into the liquid and moved into the interface, afterwhich measurements were taken. For spray-dried DSPC particle analysis,measurements for DI water were made and confirmed to be 72±1 mN/m. Theglassware and plate were re-cleaned if the surface tension was notwithin expectation. Approximately 0.5 mg of dry DSPC crystal wassprinkled carefully onto the surface while the plate was dipped into theDI water. Measurements were started immediately after the powder wasadded. Care was taken to ensure dry powder did not adsorb to the plate.Measurements were ceased if any powder had contacted the plate surface.The equilibrium surface tension of distearoylphosphatidylcholine (DSPC)is ca. 22 mN/m. Aqueous based DSPC liposomes adsorbed very slowly at theair/water interface as evidenced by the fact that after 240 sec., thesurface tension has not been significantly reduced. The slow adsorptionfor liposomes is due to the slow molecular diffusion of DSPC through thewater phase, resulting from its extremely low solubility in water.Surprisingly, the adsorption of DSPC in the form of spray-dried DSPCparticles is very fast, reducing the surface tension to equilibriumvalues within a few seconds. Moreover the inclusion of calcium ions hadno effect on the spreading of surfactant properties of the DSPCparticles. This rapid spreading and reduction of surface tension isindicative of what would likely occur upon contacting the spray-driedphospholipid particles with a wetted pulmonary membrane. Specifically,the present example provides a model for the effective delivery ofsynthetic lung surfactants and drugs to the lung.

Example IX Preparation of Nicotine Bitartrate Particles for pMDIs bySpray-Driving

Hollow porous nicotine bitartrate particles were prepared by aspray-drying technique with a B-191 Mini Spray-Drier (Büchi, Flawil,Switzerland) under the following spray conditions: aspiration: 80%,inlet temperature: 85° C.; outlet temperature: 56° C.; feed pump: 2.3mL/min; air flow: 28 SCFM. The feed solution was prepared by mixing twosolutions A and B immediately prior to spray drying.

Solution A: 5.2 g of hot water (T=50-60° C.) was used to dissolve 0.60 gof nicotine bitartrate (Sigma Chemicals, St. Louis Mo.), 0.127 g d-1lactose (Sigma Chemicals, St. Louis Mo.), and 90 mg calcium chloridedihydrate (Fisher Scientific, Fair Lawn, N.J.).

Solution B: A fluorocarbon-in-water emulsion stabilized by phospholipidwas prepared in the following manner. The phospholipid, 0.69 g SPC-3(Lipoid KG, Ludwigshafen, Germany) was dispersed in 29 g of hotdeionized water (T=60 to 70° C.) using an Ultra-Turrax mixer (modelT-25) at 8000 rpm for 2 minutes (T=60-70° C.). 30.2 g of perfluorooctylethane (F-Tech, Japan) was added dropwise during mixing. After thefluorocarbon was added, the emulsion was mixed for a period of not lessthan 5 minutes at 10000 rpm. The resulting coarse emulsion was thenpassed through a high pressure homogenizer (Avestin, Ottawa, Canada) at18,000 psi for 5 passes.

Solutions A and B were combined and fed into the spray-dryer under theconditions described above. A free flowing white powder was collected atthe cyclone separator. The geometric diameter of the nicotine bitartrateparticles was confirmed by laser diffraction (Sympatech Helos H1006,Clausthal-Zellerfeld, Germany), where a volume weighted mean diameter(VMD) of 2.60 μm was found. Scanning electron microscopy (SEM) analysisshowed the powders to be spherical and porous. Differential scanningcalorimetry analysis of the dry particles (TA Instruments) revealed theTm for the nicotine bitartrate in the powder to be 62° C., which issimilar to what is observed for spray-dried neat material.

Example X Preparation of Phospholipid-Based Particles ContainingNicotine Bitartrate by Spray-Drying

Hollow porous nicotine bitartrate particles were prepared by aspray-drying technique with a B-191 Mini Spray-Drier (Büchi, Flawil,Switzerland) under the following spray conditions: aspiration: 80%,inlet temperature: 85° C.; outlet temperature: 57° C.; feed pump: 2.3mL/min; total air flow: 22.4 SCFM.

A fluorocarbon-in-water emulsion stabilized by phospholipid was firstprepared. The phospholipid, 0.45 g SPC-3 (Lipoid KG, Ludwigshafen,Germany), was homogenized in 30 g of hot deionized water (T=60 to 70°C.) using an Ultra-Turrax mixer (model T-25) at 8000 rpm for 2 (T=60-70°C.). 15 g of perfluorooctyl ethane (F-Tech, Japan) was added dropwise ata rate of approximately 1-2 ml/min during mixing. After the fluorocarbonwas added, the emulsion was mixed for a period of not less than 4minutes. The resulting coarse emulsion was then processed through a highpressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5passes.

The emulsion was decanted into a beaker containing 8 mg sodium phosphatemonobasic (Spectrum Chemicals, Gardena, Calif.) and 90 mg calciumchloride dihydrate (Fisher Scientific, Fair Lawn, N.J.). The emulsionwas allowed to stir for approximately 5 min. The emulsion was thendecanted into a beaker containing 0.225 g nicotine bitartrate (SigmaChemicals, St. Louis Mo.) and was stirred for 5 minutes. The feedsolution was fed into the spray-dryer under the conditions describedabove. A free flowing white powder was collected at the cycloneseparator. The nicotine bitartrate particles had a volume-weighted meanaerodynamic diameter of 1.47 μm as determined by a time-of-flightanalytical method (Aerosizer, Amherst Process Instruments, Amherst,Mass.). The geometric diameter of the nicotine bitartrate particles wasdetermined by laser diffraction (Sympatech Helos H1006,Clausthal-Zellerfeld, Germany), where a volume weighted mean diameter(VMD) of 2.95 μm was found. Scanning electron microscopy (SEM) analysisshowed the powders to be spherical and highly porous. Differentialscanning calorimetry analysis of the dry particles (TA Instruments)revealed the Tm for the nicotine bitartrate in the powder wasapproximately 85° C.

This foregoing example illustrates the ability of the present powderengineering technology to effectively modulate the Tm throughformulation changes

Example XI

The preparation of lung surfactant powders with and without the use ofblowing agents was investigated. The resultant powders werecharacterized as to aerosol properties.

Preparation of Powders

The annex solutions were prepared by mixing calcium or sodium chloride,cetyl alcohol, tyloxapol (Sigma), and Infasurf (ONY Inc.) as describedin Table IV in a 20 ml glass vial to which was added an amount of hotdeionized water (70° C.) (approximately 0.54 g of sodium chloride wereused instead of calcium chloride in lots 1843-HS-03 and 04). The mixturewas vortexed until all solids were fully dissolved. One lot, 1843-HS-04used 200 ml ethanol as a solvent.

The emulsions were prepared by adding DPPC into a beaker to which wasadded an amount of 70° C. deionized water. The mixture was mixed in amixer on low speed for approximately 2-3 minutes. When a blowing agentwas used, PFOE was weighed out into a small flask and added dropwiseinto the DPPC/water mixture. The PFOE was added slowly over the courseof 1-2 minutes and the mixture was then allowed to continue mixing foran additional 1-2 minutes. Emulsion details are listed in Table IV.

When a blowing agent was used, the DPPC/water/PFOE mixture was thenimmediately removed from the mixer and run through a homogenizer fourtimes at 10,000-13,000 psi. The sample was then run through ahomogenizer a fifth time at 13,000-17,000 psi.

The annex solution was then added to the DPPC/water or DPPC/water/PFOEemulsion with continued stirring on a hot plate set to the lowesttemperature. The mixtures were kept at approximately 50° C. during spraydrying, which was done at the conditions listed in Table V.

TABLE IV Annex/Emulsion Formulation and Spray Drying Conditions AnnexSolution Hot* Water Atom. Out Emulsion Calcium Cetyl DI (g), Press.Temp, Flow rate DPPC PFOE Hot* DI Chloride Alcohol Water Infasurf FromLot # (psi) C. (ml/min) (g) (g) Water (g) (g) Tyloxapol (g) (g) (g) (g)Infasurf 1843-HS-01 60 60 2.5 1.560 18.5 40.42 0.144 0.120 0.174 201843-HS-03 60 58 2.5 1.234 18.5 50 0.092 0.138 10 1843-HS-04 70 43/445.0 1.236 50 (cold) 0.093 0.138 1843-HS-26 70 60 5.0 1.554 180 0.1450.120 0.176 20 1843-HS-35 56 59 2.5 0.937 14.0 35 0.043 0.420 121843-HS-38 60 55 2.5 0.227 8.0 10 0.044 0.630 18 1843-HS-50 56 57 2.51.555 180 0.145 0.128 0.177 20 1843-HS-51 50 1.5 1.165 130 0.109 0.0920.132 20 1843-HS-55 50 50 1.5 0.937 140 0.043 0.420 12 1843-HS-64 50 501.5 0.230 88.2 0.044 0.630 18 1843-HS-67 50 43 1.5 0.091 78 0.091 0.42012 1843-HS-69 70 60 5.0 0.092 48 0.091 0.420 12 1843-HS-70 50 35 1.50.090 48 0.090 0.420 12 1843-HS-77 60 43 2.5 1.540 18.2 50 0.146 101843-HS-78 50 35 1.5 0.150 48 0.030 0.420 12 1876-HS-88 60 44 2.5 0.7759.2 25 0.080 0.065 0.087 5 1876-HS-90 70 60 5.0 0.775 9.3 25 0.088 0.0640.088 5 1876-HS-92 60 60 2.5 0.776 9.3 25 0.072 0.060 0.087 5 1959-HS-3660 59 2.5 0.227 8.0 10 0.043 0.630 18 1959-HS-39 70 57 5 0.200 8.0 100.075 0.630 18 1959-HS-50 60 60 2.5 0.200 8.0 10 0.074 0.630 181959-HS-51 70 57 5 0.212 9.0 60.35 0.074 0.630 18

TABLE V Aerosol Characteristics Lipid: Fill % % % % % % < 3.3 aero- MVDLot # Yield CaCl Weight Moisture ED SD RSD Left Collected MMAD mm sizer(SYMPA) 1843-HS-01 43.0 2 2.2 4.453 86.7 2.15 2.5 2.00 90.81 3.43 461.287 2.83 1843-HS-03 36.0 No 2.2 2.205 10 3.28 32.7 17.98 16.80 5.5 14CaCl 1843-HS-04 19.0 No 5.0 1.235 7.3 2.18 30.0 30.54 10.89 — — CaCl1843-HS-26 35.4 2 5.0 5.563 66.26 7.15 10.8 4.05 69.16 3.65 441843-HS-26 35.4 2 2.2 — 82.44 4.11 5.0 11.07 92.70 2.90 57 — 1843-HS-3536.5 5.78 2.2 3.722, 2.89KF 81.63 3.04 3.7 3.9 84.98 4.25 29 2.683/2.9803.84/3.89 1843-HS-38 29.0 3.57 2.2 3.04 83.35 2.93 3.5 3.25 86.16 4.1433 2.799 3.93 1843-HS-50 27.3 2 not — — — — — — — — — — filled1843-HS-51 40.7 2 2.2 3.344 85.77 2.83 3.3 9.10 94.37 3.22 51 1843-HS-5140.7 2 5.0 — 76.09 5.33 7.0 4.19 79.41 3.85 42 — 1843-HS-55 58.0 5.782.2 2.058 67.87 10.67 15.7 5.66 71.85 3.45 46 1843-HS-64 11.5 1.79 not —— — — — — — — — — filled 1843-HS-67 27.8 1 not — — — — — — — — — —filled 1843-HS-69 11.6 1 not — — — — — — — — — — filled 1843-HS-70 40.41 2.2 3.254 54.55 5.05 9.3 8.29 59.54 3.51 45 1843-HS-78 52.0 3.40 2.22.237, 2.89KF 69.87 11.72 16.8 2.78 71.77 3.74 39 3.07 3.98 1876-HS-8851.0 3.57 2.2 5.050 83.71 8.69 10.4 7.47 90.45 3.57 45 2.76/2.80/ 2.741876-HS-90 52.8 3.57 2.2 4.871 91.88 4.15 4.5 6.87 98.67 2.94 562.16/2.16 1876-HS-92 47.3 3.57 2.2 5.066 93.31 2.58 2.8 5.13 98.40 3.2850 2.32/2.26 1959-HS-36 39.4 3.57 2.2 2.828 69.36 10.77 15.5 2.69 71.293.73 41 — 2.94/2.86 1959-HS-39 72.8 2 2.2 83.52 3.06 3.7 3.33 86.46 3.6343 — 2.68/2.67 1959-HS-50 23.3 2 not 3.150 — — — — — — — — — filled1959-HS-51 72.7 2 2.2 4.954 85.19 1.66 2 2.11 83.5 3.71 43 — 2.36/2.44

Example XII Leuprolide Acetate Particles

A single feed solution is prepared under defined conditions. The feedsolution is comprised of leuprolide acetate in the aqueous phase of afluorocarbon-in-water emulsion. The emulsion composition is listed inTable VI below. Accordingly, DSPC and calcium chloride dihydrate aredispersed in approximately 400 mL SWFI (T=60-70 C) using an Ultra-TurraxT-50 mixer at 800 rpm for 2 to 5 minutes. The perflubron is then addeddrop wise during mixing. After the addition is complete, the emulsion ismixed for an additional period of not less than 5 minutes at 10,000 rpm.The resulting coarse emulsion is then homogenized under high pressurewith an Avestin C-5 homogenizer (Ottawa, Canada) at 19,000 psi for 5discrete passes. The emulsion is transferred to the Potent MoleculeLaboratory for Leuprolide Acetate addition and spray drying.

TABLE VI Leuprolide Acetate Emulsion Composition Emulsion ComponentsAmount (grams) % solids DSPC 7.33 73% Calcium Chloride 0.67  7%Perflubron 200 NA SWFI 400 NA Leuprolide Acetate 2.00 20%Aerosol Data:

Deposition analysis is performed using a multi-stage liquid impinger(MSLI). The apparatus consists of four concurrent stages and a terminalfilter, each containing an aliquot of appropriate solvent for LeuprolideAcetate analysis. Deposition and emission data is reported in Table VIIbelow.

TABLE VII Leuprolide Acetate Aerosol Data Lot# XB2316 Device TurbospinFlow Rate 60 Lpm Emitted Dose 96% n = 20 MMAD 2.40 S4-Filter 70% n = 4

Example XIII PTH Feed Solution Preparation

A single feed solution is prepared under defined conditions. The feedsolution is comprised of parathyroid hormone in the aqueous phase of afluorocarbon-in-water emulsion. The emulsion composition is listed inTable VIII below. Accordingly, DSPC and calcium chloride dihydrate aredispersed in approximately 40 mL SWFI (T=60-70 C) using an Ultra-TurraxT-50 mixer at 8000 rpm for 2 to 5 minutes. The perfluorooctylethane isthen added drop wise during mixing. After the addition is complete, theemulsion is mixed for an additional period of not less than 5 minutes at10,000 rpm. The resulting coarse emulsion is then homogenized under highpressure with an Avestin C-5 homogenizer (Ottawa, Canada) at 19,000 psifor 5 discrete passes. The active drug is added to the emulsion andsubsequently spray dried after mixing for a period of not less than 10minutes.

TABLE VIII Parathyroid Hormone Emulsion Composition Emulsion ComponentsAmount (grams) % solids DSPC 0.825 82.5% Calcium Chloride 0.075  7.5%Perfluorooctylethane(PFOE) 28 NA SWFI 40 NA Parathyroid Hormone 0.100  10%Aerosol Data:

Deposition analysis is performed using an Anderson Cascade Impactor. Theapparatus consists of seven concurrent stages and a terminal filter.Aerosol deposition is measured gravimetrically and is reported in TableIX below.

TABLE IX Parathyroid Hormone Aerosol Data Lot# 2193-1 Device TurbospinFlow Rate 30 Lpm MMAD 2.67 S4-Filter 59% n = 2

Example XIV Preparation of Metered Dose Inhalers Containing NicotineBitartrate Particles

50 mg of nicotine bitartrate particles prepared in Examples IX, and Xwere weighed into 10 ml aluminum cans, crimp sealed a DF30/50 RCU-20cs50 μl valve (Valois of America, Greenwich, Conn.) and charged withHFA-134a (DuPont, Wilmington, Del.) propellant by overpressure throughthe stem. A Pamasol (Pfaffikon, Switzerland) model 2005 small scaleproduction plant complete with a model 2008 propellant pump was used forthis purpose. The amount of the propellant in the can was determined byweighing the can before and after the fill. The final powderconcentration in propellant was 0.5% w/w and formulated to provide anapproximate emitted dose of 110 μg nicotine bitartrate.

Example XV Andersen Impactor Test for Assessing Nicotine Bitartrate pMDIPerformance

The MDIs were tested using commonly accepted pharmaceutical procedures.The method utilized was compliant with the United State Pharmacopeia(USP) procedure (Pharmacopeial Previews (1996) 22:3065-3098)incorporated herein by reference. After 5 waste shots, 20 doses from thetest pMDIs were actuated into an Andersen Impactor.

Extraction procedure. The extraction from all the plates, inductionport, and actuator were performed in closed containers with anappropriate amount of methanol:water (1:1, v/v). The filter wasinstalled but not assayed, because the polyacrylic binder interferedwith the analysis. The mass balance and particle size distributiontrends indicated that the deposition on the filter was negligibly small.

Quantitation procedure. Nicotine bitartrate was quantitated by measuringthe absorption at 258 nm (Beckman DU640 spectrophotometer) and comparedto an external standard curve with the extraction solvent as the blank.

Calculation procedure. For each MDI, the mass of the drug in the stem(component −3), actuator (−2), induction port (−1) and plates (0-7) werequantified as described above. The Fine Particle Dose and Fine ParticleFraction was calculated according to the USP method referenced above.Throat deposition was defined as the mass of drug found in the inductionport and on plates 0 and 1. The mean mass aerodynamic diameters (MMAD)and geometric standard diameters (GSD) were evaluated by fitting theexperimental cumulative function with log-normal distribution by usingtwo-parameter fitting routine. The results of these experiments arepresented in subsequent examples.

Example XVI Andersen Cascade Impactor Results for Nicotine BitartratepMDI Formulations

The results of the cascade impactor tests for the nicotine bitartratepMDIs prepared according to Example XIV are shown below in Table X.

TABLE X Nicotine Bitartrate pMDIs MMAD Fine particle Fine Particle (GSD)μm fraction, % Dose, μg Nicotine/SPC- 3.6 70 74 3/CaCl₂/Lactose (2.0)Nicotine/SPC- 3.0 73 80 3/CaCl₂/ (1.9) NaPhosphate

Both pMDI preparations were observed by visual inspection to haveexcellent suspension stability, where little or no creaming orsedimentation occurred over 1 hour. The lactose containing formulationshad a slightly larger MMAD and lower FPF and FPD as compared with thesodium phosphate formulation. The reduction in aerosol performance forthe lactose formulation could be due to increased water content asevidenced in the reduced Tm.

The invention has now been described in detail for purposes of clarityand understanding. However, it will be appreciated that certain changesand modifications may be practiced within the scope of the appendedclaims.

We claim:
 1. A method for producing a phospholipid particulatecomposition comprising: adding a polyvalent cation to a formulationcomprising a phospholipid wherein a molar ratio of polyvalent cation tophospholipid is sufficient to increase a gel-to-liquid crystaltransition temperature of the particles compared to particles withoutthe polyvalent cation such that the particles have a gel-to-liquidcrystal transition temperature that is greater than room temperature byat least 20 C; and drying said formulation to form a dry particulatecomposition comprising hollow and porous particles, wherein said dryparticulate composition is characterized by being deliverable at anemitted dose of at least 54.55%.
 2. A method according to claim 1wherein the particulate composition comprises particles having a massmedian diameter of less than 20 microns.
 3. A method according to claim2 wherein the mass median diameter is 0.5-5 microns.
 4. A methodaccording to claim 1 wherein the particles comprise a mass medianaerodynamic diameter of less than 10 microns.
 5. A method according toclaim 4 wherein the aerodynamic diameter is 0.5-5 microns.
 6. A methodaccording to claim 1 wherein the drying step is performed by spraydrying.
 7. A method according to claim 6 wherein the spray dryingprocess comprises making a feedstock comprising the phospholipid.
 8. Amethod according to claim 7 wherein the feedstock comprises a colloidalsolution or suspension.
 9. A method according to claim 8 wherein thepolyvalent cation is added to the feedstock at a molar ratio ofcation:phospholipid of 0.25-1.0.
 10. A method according to claim 9wherein the polyvalent cation is added to the feedstock as calciumchloride.
 11. A method according to claim 1 wherein the formulationfurther comprises an active agent.
 12. A method according to claim 11wherein the active agent is a therapeutic agent.
 13. A method accordingto claim 12 wherein the therapeutic agent is selected from the groupconsisting of an antibiotic, antibody, antiviral agent, anti-epileptic,analgesic, anti-inflammatory agent and bronchodilator.
 14. A methodaccording to claim 13 wherein the therapeutic agent is insulin,calcitonin, erythropoietin (EPO), Factor VIII, Factor IX, ceredase,cerezyme, cyclosporine, granulocyte colony stimulating factor (GCSF),alpha-1 proteinase inhibitor, elcatonin, granulocyte macrophage colonystimulating factor (GMCSF), growth hormone, human growth hormone (hGH),growth hormone releasing hormone (GHRH), heparin, low molecular weightheparin (LMWH), interferon alpha, interferon beta, interferon gamma,interleukin-2, luteinizing hormone releasing hormone (LHRH), leuprolide,somatostatin, somatostatin analogs including octreotide, vasopressinanalog, follicle stimulating hormone (FSH), immunoglobulin, insulin-likegrowth factor, insulintropin, interleukin-1 receptor antagonist,interleukin-3, interleukin-4, interleukin-6, macrophage colonystimulating factor (M-CSF), nerve growth factor, parathyroid hormone(PTH), thymosin alpha 1, IIb/IIIa inhibitor, alpha-1 antitrypsin,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyribonuclease (Dnase),bactericidal/permeability increasing protein (BPI), anti-CMV antibody,interleukin-1 receptor, 13-cis retinoic acid, nicotine, nicotinebitartrate, gentamicin, ciprofloxacin, amphotericin, amikacin,tobramycin, pentamidine isethionate, albuterol sulfate, metaproterenolsulfate, beclomethasone dipropionate, triamcinolone acetamide,budesonide acetonide, ipratropium bromide, flunisolide, fluticasone,fluticasone propionate, salmeterol xinofoate, formeterol fumarate,cromolyn sodium, ergotamine tartrate and analogues, agonists andantagonists thereof.
 15. A method according to claim 13 wherein thetherapeutic agent is tobramycin.
 16. A method according to claim 13wherein the therapeutic agent is ciproflaxcin.
 17. A method according toclaim 13 wherein a formulation bulk density is less than 0.05 g/cm³. 18.A method according to claim 13 wherein the drying step comprises spraydrying, and wherein an inlet temperature is between 60° C. and 170° C.and an outlet temperature is about 40° C. to 120° C.
 19. A methodaccording to claim 13 wherein the therapeutic agent is amphotericin.